Hyperoside regulates its own biosynthesis via MYB30 in promoting reproductive development and seed set in okra

Abstract

Flavonoids are secondary metabolites that play important roles in fruit and vegetable development. Here, we examined the function of hyperoside, a unique flavonoid in okra (Abelmoschus esculentus), known to promote both flowering and seed set. We showed that the exogenous application of hyperoside significantly improved pollen germination rate and pollen tube growth by almost 50%, resulting in a 42.7% increase in the seed set rate. Of several genes induced by the hyperoside treatment, AeUF3GaT1, which encodes an enzyme that catalyzes the last step of hyperoside biosynthesis, was the most strongly induced. The transcription factor AeMYB30 enhanced AeUFG3aT1 transcription by directly binding to the AeUFG3aT1 promoter. We studied the effect of the hyperoside application on the expression of 10 representative genes at four stages of reproductive development, from pollination to seed maturity. We firstly developed an efficient transformation system that uses seeds as explants to study the roles of AeMYB30 and AeUFG3aT1. Overexpression of AeMYB30 or AeUF3GaT1 promoted seed development. Moreover, exogenous application of hyperoside partially restored the aberrant phenotype of AeUF3GaT1 RNA-interference plants. Thus, hyperoside promotes seed set in okra via a pathway involving AeUF3GaT and AeMYB30, and the exogenous application of this flavonoid is a simple method that can be used to improve seed quality and yield in okra.

The flavonoid hyperoside promotes reproductive development and improves seed set in okra via a positive feedback mechanism.

Introduction

Okra (Abelmoschus esculentus L. Moench.) is a traditional vegetable crop of the Malvaceae family, which has been cultivated for over 3,000 years. It is currently grown all over the world with 68 million hectares of dedicated arable land and 200 cultivated varieties (Purseglove, 1988; De Carvalho et al., 2011). It is mainly grown for its edible leaves but can also be used for its fruit, as a staple food, vegetable, and for medicine (Arapitsas, 2008). However, okra flowers bloom and wilt over the course of a single day, like other Malvaceae family include cotton (Gossipium sp.), cacao (Theobroma cacao), and hibiscus (Hibiscus rosa-sinensis; Jain et al., 2012). Therefore, the low seed setting rate of okra caused by short flowering resulted in the decrease of yield.

High seed setting rate depends on the stable and sufficiently sexual reproduction. Only with good development of female tissue of the pistil and the pollen tube of the male gametophyte, can we have full, high quality and yield seeds. Before petals fall successful communication between the pistil and the pollen tube results in fruit and seed formation and three major events take place: (1) a pollen grain (male gametophyte) lands on and adheres to the stigma; (2) pollen tubes are guided through the female reproductive tissues to the ovule via the micropyle; (3) pollen tubes need to be properly attracted and guided to reach the ovule for fertilization to happen (Chen et al., 2002; Zhong et al., 2019). Pollen tube growth is rapid and polarized and occurs exclusively at the tip. The contact of a pollen grain with the stigma initiates a signal network based on Rop GTPases that controls the growth of the pollen tube tip. Flavonoids have been reported to stimulate the development, germination and growth of pollen tubes in tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), and petunia (Petunia hybrida) during in vitro culture assays (Ylstra et al., 1992; Muhlemann et al., 2018). Rop GTPases may be directly activated by the Rop guanine nucleotide exchange factor RopGEF (Ylstra et al., 1992; Muhlemann et al., 2018). When pollen tubes grow rapidly forward before fertilization, actin depolymerizing factors (ADFs) are the main factor controlling actin depolymerization and are also the limiting factor in rapid actin turnover (Daher and Geitmann, 2012). ADFs have been implicated in numerous physiological processes not limited to pollen tube growth or pollen germination but also including flowering time, plant defense, and root hair growth (Jiang et al., 1997; Dong et al., 2001; Gilliland et al., 2003). Small cysteine-rich proteins (CRPs, also called LUREs) are secreted by the female gametophyte to guide and direct pollen tube growth (Dresselhaus and Marton, 2009; Higashiyama, 2010).The female gametophyte also accumulates receptor kinases at the plasma membrane, for example FERONIA. In its absence, pollen tube growth fails to rupture even after it has reached the female oocyte and continues to grow, or the pollen tube breaks or dies prematurely (Tsukamoto et al., 2010; Kessler and Grossniklaus, 2011).

Flavonoids are polyphenolic compounds that are widespread in the plant kingdom and play a central role in fruit development (Bogs et al., 2006; Tohge et al., 2013; Tordesillas et al., 2017). For example, six anthocyanidins (pigments that belong to the flavonoid class of molecules) generally accumulate in fruits and have recognized health benefits. Anthocyanidins are normally bound to one or more sugar moieties (Jaakola, 2013). Flavonoids can be classified into multiple families (flavanols, flavanones, isoflavones, flavonols, and anthocyanidins; Tohge et al., 2017; Nabavi et al., 2018) and play different roles in plant growth and fruit development (Kumar and Pandey, 2013). Flavonoids are derived from the phenylalanine pathway: their synthesis starts with the formation of 4-coumaroyl-CoA via phenylalanine ammonia lyase, cinnamate 4-hydroxylase (C4H), and 4-coumaroyl CoA ligase (4CL). Chalcone synthase (CHS) ultimately shunts the phenolic compounds into the flavonoid biosynthesis pathway. Chalcone isomerase (CHI) and flavanone 3 β-hydroxylase (F3H) form flavone as intermediate products, which are subsequently converted by a variety of enzymes into different flavonoid subfamilies that share a similar structure and may therefore share a similar function (Fossen et al., 1999; Ravaglia et al., 2013; Brodowska, 2017). Glycosylation of flavonoids plays an important role not only in the downstream steps of their biosynthetic pathway, but also in the stabilization of hydrophobic flavonoids, by way formation of an external hydrogen bond between the sugar and water molecules (Fossen et al., 1999; Ravaglia et al., 2013). Different varieties of okra extracts are rich in the flavonoid hyperoside whose pharmacological activity has been well studied (Wu et al., 2007; Zhou et al., 2012). Hyperoside, also known as quercetin 3-O-β-d-galactopyranoside, is a flavonol glycoside compound composed of two phenyl rings (A and B rings), a six-membered oxygen heterocycle (C ring), and a galactopyranoside (Wang and Yang, 1981). The biosynthetic pathway of hyperoside in okra was reported by our previous study (Yang et al., 2020). The enzyme UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GaT) catalyzes this glycosylation step onto the 3-O-position of flavonol and constitutes the last step in hyperoside biosynthesis (Fossen et al., 1999; Ravaglia et al., 2013). The genes encoding the enzymes for the entire flavonoid biosynthetic pathway are under tight transcriptional regulation by the MYB-bHLH-WD40 (MBW) complex (Serna, 2007). However, the MBW complex is not the only group of TFs playing a major role in flavonoid regulation, for example AtMYB11 and 12 in Arabidopsis are important for the regulation of flavonols, while the MBW complex regulates anthocyanin and proanthocyanidins (PAs; Stracke et al., 2007, 2010). Although many genes involved in the MBW complex have been identified, gaps remain in precisely how they regulate this biosynthetic pathway, especially concerning the role of MYB domain-containing proteins. MYB proteins belong to a large family of transcription factors and are key regulators of the biosynthesis of flavonoid compounds (Serna, 2007).

Flavonoids are widely studied as developmental regulators of plant reproduction, for instance during flower formation and opening, pollen tube growth, embryo development, fruit, seed formation, and maturity (Broun, 2005; Taylor and Grotewold, 2005). The importance of flavonoids is underscored by the observation that higher flavonoid content tends to be associated with higher seed set and fruit quality, even when no limits are imposed on pollination or fertilization (Ylstra et al., 1992; Muhlemann et al., 2018). For example, anthocyanin-type flavonoids largely accumulate during the fruit ripening phase and are thought to provide health benefits (He and Giusti, 2010). As plant growth regulators and protectants, small molecules metabolites can improve the tolerance of edible plants to biotic and abiotic stress (Meng et al., 2018b; Zhao et al., 2019). Flavonoids protect plants from a variety of biological and abiotic stresses, which are caused by environmental stresses such as pathogen attack during seed development (Treutter, 2006; Pourcel et al., 2007). Furthermore, the mechanism of flavonoids to help seeds resist adversity, promote seed development and improve seed quality is likely to be the protective effect of flavonoids on seeds in terms of flavonoid oxidation (Porter and Wareing, 1974; Yamasaki et al., 1997). In the process of seed development, an important flavonoid, catechin reacts with kaempferol to yield heterodimers determining the senescence and oxidation of seed (Jiang et al., 2004). Flavonoids accumulated in different crop seeds underlie seed coat browning and water-impermeability enabling better germination or allowing for higher quality seeds in order to get better germination or edibility or industrial oil extraction (Halloin, 1982; Egley et al., 1983). In addition to flavonoids, other compounds such as sorbitol as signal molecules have been found to regulate plant reproductive development processes (Meng et al., 2018a; Li et al., 2020b). Okra is rich in the flavonol hyperoside; most studies have focused on separating the various compounds present in okra extracts due to their antioxidant and potential anti-inflammatory classified properties (Shen et al., 2019). It has been reported that small metabolites play an important role in the development of apple fruit quality (Li et al., 2020a). It would be desirable, although rarely reported, to use endogenous hyperoside as a growth regulator to improve the efficiency of pollination fertilization and seed set, with the ultimate goal to improve fruit yield and quality. However, the relationship between hyperoside and seed set is unknown in okra.

Research in nonmodel plant systems like okra suffers from the lack of efficient and stable transgenic procedures. It is therefore crucial to establish an efficient procedure for the generation of transgenic okra. In this study, we developed a fast and efficient transgenic system that uses seed embryos as explants, as a first step towards characterizing hyperoside biosynthesis and the effect of this flavonoid on seed development and seed quality. In different phases from pollen germination to seed development, we screened several genes that were positively induced by hyperoside treatment during different developmental phases, from pollen germination to seed maturation: the okra gene AeUF3GaT1, encoding a UDP-glucose, flavonoid 3-O-glucosyltransferase, was the most upregulated. The overexpression of AeUF3GaT1 in transgenic okra plants promoted pollen germination, pollen tube growth, and ultimately ameliorated seed set, along with an increase in flavonoids and hyperoside content. Moreover, exogenous application of hyperoside partially restored the phenotypes exhibited by AeUF3GaT1 RNA-interference lines, supporting the role of hyperoside. The successful establishment of a transgenic system for okra will facilitate research exploring the mechanisms linking AeUF3GaT1, hyperoside, and seed set.

Results

Hyperoside content gradually increases during fruit development in okra

As okra is self-compatible, we used anthesis as a visible marker to synchronize fruit development and the associated dynamic changes. Although the fruit epidermis was smooth 2 d after anthesis (DAA2), glandular hairs began to grow on the fruit epidermis at DAA4, marking the beginning of fruit maturation. On DAA16, fruits underwent a sudden expansion (Figure 1A). Between DAA2 and DAA20, we measured the following fruit traits: average maximum width, average length, and average weight per fruit. All of these indicators increased abruptly on DAA16 (Figure 1B).

Figure 1.

Changes in developing okra fruit from 2 to 20 DAA. A, Fruit appearance during development from 2 to 20 DAA. B, Average maximum width, average length, and average weight per fruit of developing fruits from 2 to 20 DAA. C, Total flavonoid content of developing fruits from 2 to 20 DAA. FW, fresh weight. D–I, The content of hyperoside and five other flavonoids, rutin, isoquercetin, myricetin, quercetin-3-O-gluc, and quercetin, in developing fruits from 2 to 20 DAA. FW, fresh weight. In B–I, error bars show the standard error of the mean (SEM; n = three biological replicates)

We next determined the content of total flavonoids in the fruit by high-performance liquid chromatography (HPLC) and detected a gradual increase over the course of the fruit ripening process (Figure 1C). Among the six main flavonols detected (rutin, isoquercetin, hyperoside, myricetin, quercetin-3-O-glucoside, and quercetin), hyperoside accumulated throughout fruit development and increased sharply at DAA16. We therefore selected fruits and seeds at DAA16 to further study the effects of hyperoside treatment (Figure 1, DG).

Hyperoside application increases fruit size and seed set and promotes flavonoid accumulation in okra seeds

To establish the effect of hyperoside on okra fruit development, we periodically sprayed plants with 50 mg·L^–1 of hyperoside or with each of the other five flavonols detected in okra before flowering (Supplemental Figures S1–S7). Only the hyperoside treatment had an effect on fruit or seed morphology (Supplemental Figure S7). We measured changes in the morphology of the epidermis (external and internal) among the following three test groups: plants that had not been sprayed, plants sprayed with buffer alone, and plants sprayed with hyperoside. Okra fruits develop a coat covered with tiny thorn, also called a “fruit thorn”, that is an important factor in the commodity quality of okra fruit. Scanning electron microscopy (SEM) revealed a mature peltate coat structure in all three test groups, with both small and large fruit thorns. Small fruit thorns were evenly arranged in rows in the two control groups but were more numerous in the hyperoside-treated group. We observed no significant differences in the developed large fruit thorns or the internal epidermis among the three groups (Supplemental Figure S6).

Although hyperoside treatment significantly increased fruit size, it did not affect pericarp development. Likewise, the outer seed epidermis of all three groups exhibited a similar pattern of surface hairs, which resemble developing small fruit thorns and are arranged in columns (Supplemental Figure S7, A and B). We cut seeds longitudinally to observe the upper part of the seed kernel and the seed coat border (Supplemental Figure S7, B and C; magenta squares) as well as the center of the seed kernel (blue squares). The seed kernel and coat were clearly more closely connected in the hyperoside treatment group, whereas fat particles in the middle of the seed kernel had the same shape in all three groups (Supplemental Figure S7C).

We determined the average single fruit weight, average fruit length, and average maximum fruit width at DAA16 in all three groups. All of these measurements were higher in plants sprayed with hyperoside than in the controls (Figure 2, A and B). We also measured seed establishment and seed set in DAA16 fruits that had been dissected lengthwise. The arrangement of seeds in the ventricles appeared more uniform and orderly in hyperoside-treated fruits, accompanied by fewer aborted seeds. Hyperoside application increased seed set to 88.9% compared with 62.3% for the no-spray control and 63.5% for the buffer-only group (Figure 2B). In agreement with this observation, the average number and weight of seeds were significantly higher in the sprayed group than in the two control groups (Figure 2B).

Figure 2.

Hyperoside application causes increased fruit size and seed set. A, Surface views and longitudinal sections of representative okra fruits from the control, buffer, and hyperoside treatment groups at 16 DAA. Bar=1 cm. B, Characteristics of fruit from the three treatment groups: average weight per fruit, fruit length, fruit width, seed set, number of seeds per fruit, weight per thousand seeds, and seed size. Error bars show the standard error of the mean (sem; n = three biological replicates). *P < 0.05 (Student’s t test). C, The contents of total protein, total fat, total polysaccharide, and total flavonoids in seeds DAA16 for the control, buffer, and hyperoside treatment groups. Different letters (a and b) indicate significant differences (P<0.05) between genotypes using Duncan’s multiple range test after analysis of variance (ANOVA). D, HPLC profiles of fruit from the three treatment groups. 1–6 indicate the reference standards: 1. Rutin, 2. Isoquercetin, 3. Hyperoside, 4. Myricetin, 5. Quercetin-3-O-glucoside, 6. Quercetin. E, The content of six flavonoids in fruit from the three treatment groups, analyzed with standards. Error bars show the standard error of the mean (sem; n = three biological replicates). *P < 0.05 (Student’s t test). FW, fresh weight

Due to the effect of hyperoside on seed establishment, we next focused on seed nutritional indicators by analyzing the total protein, fat, polysaccharide, and flavonoid contents in seeds of the no-spray, buffer-only, and hyperoside-treated plants at DAA16. As shown, there was no significant difference in the content of total protein among the three groups. The content of total fat in the hyperoside treatment group was higher than the other two groups, but the difference was not significant. There was no significant difference in the content of total polysaccharide in okra. However, the flavonoid content was significantly higher in the treated group than in the two control groups (Figure 2C). This may be because hyperoside, as a signal substance, can specifically promote the accumulation of flavonoids and reproductive development and has no significant effect on the nutritional components. We performed HPLC on seed extracts to determine the relative composition of the six main flavonols, i.e. rutin, isoquercitrin, hyperoside, myricetin, quercetin-3-O-glucoside, and quercetin. Of these, only the hyperoside content was significantly increased in the developing fruits of the treated group (Figure 2D). The hyperoside content in the seeds of the hyperoside application group was about 4.3 mg·g⁻¹ fresh weight (FW), which is twice as much as that of the no-spray control (2.1 mg·g⁻¹ FW) and buffer (2.2 mg·g⁻¹ FW) groups (Figure 2E). For a more comprehensive metabolomic analysis, we also quantified other flavonoid-related compounds in developing fruit (DAA2–20), including PAs, genistins belonging to the isoflavone family, and luteolin belonging to the flavone family. There was no significant difference in the content of these compounds between the fruits of plants in the different treatment groups (Figure 2E).

To fully characterize the effect of hyperoside on the overall seed metabolism, we analyzed the expression of several key genes involved in the phenylpropanoid biosynthesis pathway, which gives rise to flavones, isoflavonoid, flavonol, and proanthocyanidins in okra (Supplemental Figure S8A). The expression of FLAVONE SYNTHASE I (AeFNSI), ISOFLAVONE SYNTHASE (AeIFS), HOMOISOCITRATE DEHYDROGENASE (AeHIDH), DIHYDROFLAVONOL-4-REDUCTASE (AeDFR), LEUCOCYANIDIN REDUCTASE (AeLAR), LEUCOANTHOCYANIDIN DIOXYGENASE (AeLDOX), and ANTHOCYANIDIN REDUCTASE (AeANR) was not significantly induced by hyperoside treatment (Supplemental Figure S8B). Therefore, application of hyperoside before flowering affected the accumulation specifically of hyperoside, and particularly in seeds.

Hyperoside induces the expression of the flavonol biosynthetic genes AePAL2, AeCHS4, AeCHI3, AeFLS1, and AeUF3GaT1

To determine the molecular mechanism underlying the increase in flavonoid content by hyperoside treatment and improve seed set, we next analyzed the relative expression of genes encoding key putative enzymes in hyperoside biosynthesis in seeds. In a previous study, we observed that 6 out of 10 putative biosynthetic genes were upregulated in okra flowers in response to hyperoside treatment (Yang et al., 2020). Here, we observed that the transcript levels of 5 out of the 10 hyperoside biosynthetic genes were induced (marked in red in Figure 3A): AePAL2, AeCHS4, AeCHI3, AeFLS1, and AeUF3GaT1. Each gene belongs to a small gene family (three genes for PAL, two for CHS, three for CHI, five for FLS, and six for UF3GaT) but were the only members of the respective families that were upregulated under these conditions.

Figure 3.

Hyperoside upregulates the expression of genes for key enzymes in the hyperoside biosynthetic pathway. A, The hyperoside biosynthetic pathway in okra. Enzymes that are upregulated in hyperoside-treated plants are marked in red. B, The relative expression (fold change) of the key enzyme genes in the three treatment groups. The most upregulated genes in the hyperoside-treated group encode phenylalanine ammonia lyase (AePAL2), chalcone synthase (AeCHS4), chalcone isomerase (AeCHI3), flavonol synthase (AeFLS1), and UDP-galactose: flavonoid 3-O-galactosyltransferase (AeUF3GaT1). Error bars show the standard error of the mean (sem; n = three biological replicates). *P <0.05 (Student’s t test).

Out of all the upregulated genes, AeUF3GaT1 showed the greatest increase in expression following hyperoside treatment (14.3-fold over controls). AeUF3GaT1 encodes the enzyme that catalyzes the last and dedicated step in hyperoside biosynthesis (Figure 3B), making it an excellent candidate for further characterization. AeUF3GaT1 is highly similar to the Arabidopsis UGT78D1 gene (AtUF3GaT1, At1g30530; https://www.arabidopsis.org/; Supplemental Figures S9–S10).

Hyperoside promotes reproductive development and induces the expression of reproductive genes and flavonoid biosynthesis genes, especially AeUF3GaT1, during seed establishment

Whether a seed will form is closely linked to pollen germination, pollen tube growth, and fertilization. We therefore measured average pollen germination rates (APGRs) and average pollen tube length (APTL) in vitro for all three test groups as a proxy for pollen viability. We scored pollen germination after 30 min and measured pollen tube length after 1 h of culture in vitro. The APGR for the hyperoside-treated group was 69.3% compared to 46.3% for the no-spray control and 47% for the buffer-only group. Similarly, pollen tubes grew to a length of 59.1 µm in the hyperoside application group compared to 39.7 μm for the no-spray control and 41.5 μm for the buffer-only group (Figure 4A).

Figure 4.

Hyperoside specifically promotes pollen function: representative genes in the four phases from pollen germination to seed development. A–F, APGR and APTL for control plants and plants treated with buffer or one of six flavonoids (hyperoside, rutin, isoquercetin, myricetin, quercetin-3-O-gluc, and quercetin). G, Seed production can be divided into four phases: I pollen germination; II pollen tube growth; III double fertilization; and IV seed development. H, RT-qPCR analysis of the expression of 20 representative genes upregulated by hyperoside during the four phases of seed production. The genes exhibiting the most upregulation are indicated in red. I, The relative expression of AeUF3GaT1, a key gene in seed development and flavonoid biosynthesis, during Phases I–III of seed production in control, buffer, and hyperoside treatment groups. Error bars show the standard error of the mean (sem; n = three biological replicates). *P <0.05 (Student’s t test).

We also assessed the effects of adding the other flavonoids (rutin, isoquercitrin, myricetin, quercetin-3-O-glucoside, and quercetin) to the pollen culture medium. At best, these flavonoids had modest positive effects on pollen germination and pollen tube length: the APGR was 52% for rutin application, 50% for isoquercitrin, 45.7% for myricetin, 42.3% for quercetin-3-O-glucoside, and 40.6% for quercetin (Figure 4, B–F). Similarly, the APTL was 41.3 μm for the rutin treatment, 42.3 μm for isoquercitrin, 40.6 μm for myricetin, 42.7 μm for quercetin-3-O-glucoside, and 39.7 μm for quercetin (Figure 4, B–F). Thus, only hyperoside exhibited strong positive effects on pollen germination rates and pollen tube growth.

The pollen tube, a membrane-surrounded extension of the male gametophyte, carries sperm cell nuclei through the pistil to the female gametophyte to complete double fertilization. Successful fertilization depends on an active dialogue between male and female gametophytes. We divided the reproductive process from pollination to seed maturity into four phases: pollen germination (Phase I), pollen tube growth (Phase II), double fertilization (Phase III), and seed development (Phase IV; Figure 4G). Because many genes are involved in reproductive growth, we used a set of marker genes to test whether reproductive growth is induced specifically by hyperoside. We identified five representative and pollen- or seed-specific genes for each of the four phases of reproduction and measured their expression to determine the effect of hyperoside on reproductive development. The transcript levels of GMP Exchange Factor (AeGEF2), Actin Depolymerizing Factor (AeADF5), and FERONIA Recptor Protein Kinase (AeFER6) increased 10-fold in the hyperoside application group relative to the no-spray control and buffer groups, and AeUF3GaT1 was even more highly induced (15-fold over controls; Figure 4H). The relative expression of AeUF3GaT1 during Phases I–III of seed production was also somewhat upregulated by hyperoside treatment (Figure 4I).

During Phase I, the expression levels of AeROP2, a member of the Rho-like small GTPase family, and AeGEF2, a ROP guanine nucleotide exchange factor, were significantly higher in the hyperoside application group compared to the no-spray control and buffer groups. Rho-like small GTPases initiate signal transduction during pollen germination and rapid tip growth. ROP guanine nucleotide exchange factors then influence the elongation of pollen tubes. Thus, hyperoside upregulates the activity of genes known to function in pollen germination and early pollen tube growth.

During Phase II, the transcript levels of the leucine-rich repeat extensin (LRX) gene AeLRX11, the pectin methylesterase inhibitor (PMEI) gene AePME1.2, the ADF gene AeADF5, and the stomatal closure-related actin binding (SCAB) gene AeSCAB1 were higher in the pollen tubes of hyperoside-treated flowers than in the no-spray control and buffer-only groups. LRX and PMEI proteins play critical roles in the dynamic changes that underlie pollen tube development and normal growth. In the pollen tube, ADFs modulate the structure and dynamics of actin filaments, thereby influencing their higher order organization. SCABs function in the physiological processes of cell morphogenesis, cell movement, and intracellular material transport linked specifically to plant reproduction processes.

During Phase III, hyperoside application induced the expression level of AeLURE4 and AeFER6. LURE-type CRPs are secreted at the micropylar ends of synergid cells to guide short-range micropylar pollen tube growth (Zhong et al., 2019). FERONIA proteins attract pollen tubes to the synergid and induce their bursting, and hence the release of sperm cells, once they enter the female oocyte.

During Phase IV, we measured the expression levels for five flavonoid biosynthetic genes and detected upregulation of two of them (Figure 4H). AePAL2, which encodes an enzyme that acts early in hyperoside biosynthesis, was upregulated eight-fold (Figure 4H). AeUF3GaT1, encoding the UDP-galactose: flavonoid 3-O-galactosyltransferase enzyme and the last step of hyperoside biosynthesis (Maeda and Dudareva, 2012; Wang et al., 2019), displayed the strongest degree of induction (15-fold) of all genes measured in the hyperoside application group. AeUF3GaT1 was also induced during Phases I–III, although not to the same extent as during phase IV (Figure 4I). We also determined the transcript levels for these five flavonoid biosynthetic genes from Phases I to IV (DAA0–20, spanning the entire process of seed development): they were all upregulated sharply at DAA16 (Supplemental Figure S11). We further explored the molecular mechanism by which AeUF3GaT1, the gene upregulated to the greatest extent by hyperoside treatment, improves the seed setting rate.

AeMYB30 increases AeUF3GaT1 transcription and improves reproductive development, seed set, and hyperoside accumulation in transgenic okra seeds

In earlier work, we uncovered a link between the stabilization of the transcription factor AeMYB30 and enhanced transcription of AeUF3GaT1, which ultimately led to hyperoside accumulation and prolongation of the full-blooming period by 6 h (Yang et al., 2020). Considering the importance of the AeMYB30-AeUF3GaT1 module during reproductive development, we explored its potential role using our first-established transformation system that uses seed embryos as ex-plants. After about 14 d of co-culture with Agrobacterium, embryogenic calli differentiated well, at which point they were transferred to bud induction medium to produce buds about 28 d later. Buds completed their differentiation within 2 weeks and were well established after 3 weeks (or 35 d from the start of co-culture). We then transferred 3- to 5-cm buds to root induction medium, and the young root tissue differentiated over the next week. After 2 weeks of rooting, the plantlets were transferred to the greenhouse with a survival rate of 100%, where they bloomed and bore fruits generally 84 d after the start of co-culture. Finally, we harvested fruits and seeds after 100 d (Supplemental Figure S12).

Hyperoside accumulated to high levels in vivo in fruits and seeds treated with exogenous hyperoside. We therefore tested whether AeMYB30 acted on hyperoside biosynthesis at the transcriptional level. We performed a yeast one-hybrid (Y1H) assay to investigate the binding of AeMYB30 to the AeUF3GaT1 promoter (Figure 5A). We tested several promoter fragments and observed binding of AeMYB30 only to fragments containing the AeMYB30-binding site (CAACAG). We then fused the full-length AeMYB30 coding sequence to the reporter gene GFP and overexpressed this construct in transgenic okra seeds. A chromatin immunoprecipitation polymerase chain reaction assay using an antibody raised against GFP confirmed that AeMYB30 did bind to the AeUF3GaT1 promoter of AeMYB30 overexpression transgenic okra seeds in vivo (Figure 5B). We generated both RNAi lines and overexpression lines for AeMYB30 (Figure 5C) and the AeMYB30 transcript lines and overexpression lines for AeMYB30 (Figure 5C). The AeMYB30 transcript level was decreased to 20% (line 1) and 35% (line 2) in the RNAi lines, respectively, whereas it was increased to approximately eight times, relative to that in the nontransgenic control. Consequently, the AeUF3GaT1 transcript levels showed corresponding changes to those of AeMYB30 in the transgenic lines (Figure 5C). The AeMYB30 overexpression transgenic lines showed much higher pollen germination rates and pollen tube growth than the non-transgenic controls (Figure 5D). Furthermore, the AeMYB30 overexpression lines displayed a higher content of total flavonoids, particularly of hyperoside (Figure 5E).

Figure 5.

AeMYB30 promotes AeUF3GaT1 transcription, which is required for reproductive development, seed development, and hyperoside biosynthesis in seeds. A, Y1H analysis showing that AeMYB30 binds to the AeUF3GaT1 promoter fragment (ProAeUF3GaT1). The promoter of AeUF3GaT1 was divided into three fragments (P1– P3). AbA (Aureobasidin A), a yeast cell growth inhibitor, was used as a screening marker. The basal concentration of AbA was 200 ng·mL⁻¹. Rec-p53 and the P53-promoter, whose interaction has been confirmed, were used as a positive control. Empty vector and the AeUF3GaT1 (P; 1,235 bp) promoter were used as negative controls. B, ChIP-PCR showing that AeMYB30 binds to the AeUF3GaT1 promoter in vivo. Cross-linked chromatin samples were extracted from seeds of AeMYB30-GFP overexpressing transgenic okra plants and precipitated with an anti-GFP antibody. Sequences were amplified from the eluted DNA by qPCR. Four regions (S1–S4) of the AeUF3GaT1 promoter were investigated (see diagram). Seeds of transgenic okra plants overexpressing the GFP sequence were used as negative controls. The ChIP assay was repeated three times, and the enriched DNA fragments in each ChIP were used as one biological replicate for qPCR. C, The relative expression (fold change) of AeMYB30 and AeUF3GaT1 in wild-type (WT), empty vector, AeMYB30-RNAi-line1, AeMYB30-RNAi-line2, AeMYB30-OE-line1, and AeMYB30-OE-line2. D–F, Average seed set, APGR (after 1 h of culture), APTL, and the total flavonoid and hyperoside content in seeds of different transgenic lines of C. In (C)–(F), error bars show the standard error of the mean (sem; n = three biological replicates). *P <0.05 (Student’s t test)

AeUF3GaT1 overexpression significantly promotes pollen germination and pollen tube growth and increases okra seed set

To further examine the role of AeUF3GaT1, we also generated lines harboring the AeUF3GaT1 RNAi vector (AeUF3GaT1-RNAi). We characterized two RNAi lines, with AeUF3GaT1 transcript levels that were 23% (line 1) and 35% (line 2) the levels of those in the nontransgenic control plants (Figure 6B). Transgenic plants bearing the empty vector were used as the control (Figure 6, A and B). We scored seed establishment, seed set, pollen germination, and pollen tube growth in these lines and also in the AeUF3GaT1 overexpression lines at DAA16.

Figure 6.

Overexpression of AeUF3GaT1 in transgenic okra significantly promotes pollen function and increases seed set. A, Schematic diagram of constructs used for overexpression (OE) and RNA-interference (RNAi) of AeUF3GaT1. B, The relative expression (fold change) of AeUF3GaT1 in wild-type (WT), empty vector, AeUF3GaT1-RNAi-line1, AeUF3GaT1-RNAi-line2, AeUF3GaT1-OE-line1, and AeUF3GaT1-OE-line2. C–E, Average seed set, APGR (after 1 h of culture), APTL, and the total flavonoid and hyperoside content in seeds of different transgenic lines of b. In (b)–(e), error bars show the standard error of the mean (sem; n = three biological replicates). *P <0.05 (Student’s t test)

The arrangement of seeds in the ventricles was more uniform in lines overexpressing AeUF3GaT1, which also had fuller seeds and fewer aborted embryos compared to the wild-type, empty vector control, and RNAi lines (Figure 6, B and C). In addition, the APGR and APTL were significantly higher than those of the wild-type, empty vector control, and RNAi lines (Figure 6D).

We then analyzed the total flavonoid and hyperoside content in all transgenic lines and in the wild type. As expected, both total flavonoid content and hyperoside accumulated to higher levels in the overexpression lines than in the wild-type, empty vector control, and RNAi lines (Figure 6E). Transcript levels for 11 hyperoside-induced genes (AeGEF2, AeADF5, AeFER6, AeUF3GaT1, AeMYB30, AeROP2, AeLRX11, AeSCAB1, AePMEI1.2, AeLURE4, and AePAL2) were strongly upregulated in the AeMYB30 and AeUF3GaT1 RNAi lines and also in the overexpression lines relative to the wild-type and empty vector controls (Figure 7A;Supplemental Figure S13), as well as average seed set, APGR, APTL, total flavonoids, and hyperoside contents (Figure 7B).

Figure 7.

The wild-type (WT), empty vector, AeMYB30-RNAi-line1, AeMYB30-RNAi-line2, AeMYB30-OE-line1, AeMYB30-OE-line2, AeUF3GaT1-RNAi-line1, AeUF3GaT1-RNAi-line2, AeUF3GaT1-OE-line1, and AeUF3GaT1-OE-line2 in response to hyperoside. A, The four representative genes in four phases in response to hyperoside. B, The average seed set rates, APGR, APTL, and the content of total flavonoids and hyperoside in the WT, empty vector, AeMYB30-RNAi-line1, AeMYB30-RNAi-line2, AeMYB30-OE-line1, AeMYB30-OE-line2, AeUF3GaT1-RNAi-line1, AeUF3GaT1-RNAi-line2, AeUF3GaT1-OE-line1, and AeUF3GaT1-OE-line2. FW, fresh weight. In (A) and (B), error bars show the standard error of the mean (sem; n = three biological replicates). *P <0.05 (Student’s t test)

Discussion

Okra belongs to the Malvaceae family which were widely used in our daily life and various flavonoids in okra are secondary metabolites that participate in a variety of physiological functions (Winkel-Shirley, 2001). We wondered whether hyperoside might be used as a growth and development regulator, with the ultimate goal of increasing seed set and seed quality. The expression of flavonoid biosynthesis genes is significantly upregulated during ripening of apples (Malus domestica), strawberries (Fragaria sp.), and grapes (Vitis sp), leading to the accumulation of flavonoids in ripe fruits (Moyano et al., 1998; Halbwirth et al., 2006; Hu et al., 2016). However, whether the accumulation of flavonoids is the cause or the result of fruit maturation is unknown. Furthermore, it is unclear whether flavonoids might be harnessed as growth regulators to promote seed set.

Seed flavonoid contents have been used as a standard to evaluate their physiological and agronomic characteristics, especially for grain crops such as maize (Zea mays), rice (Oryza sativa), and soybean (Glycine max; Shirley, 1998). Mutants lacking flavonoids provide a useful tool for studying metabolic biosynthesis and their physiological roles. For example, flavonoid accumulation in pollen is associated with high pollen germination and seed set (Taylor and Grotewold, 2005). Inhibited expression of the flavonoid synthesis gene in Petunia anther leads to male sterility (Van Der Meer et al., 1992). Flavonols are an important class of flavonoids that promote pollen tube growth and integrity by regulating ROS homeostasis caused under high-temperature abiotic conditions in tomato (Muhlemann et al., 2018). Flavonols with a signaling function could stimulate pollen development, pollen germination, and pollen tube growth in tobacco (Ylstra et al., 1992). Therefore, analyzing the genes and enzymes participating in flavonol and hyperoside biosynthesis is a prerequisite to determine their vital role in reproductive development.

We previously performed deep sequencing of the transcriptome (RNA-seq) of okra flowers from the same three test groups used in our current study to identify the genes that encode putative key enzymes in the hyperoside biosynthesis pathway (Yang et al., 2020). In this study, we independently verified the expression patterns of these genes in seeds and established that AePAL, AeCHS, AeCHI, AeF3H, AeFLS, and AeUF3GaT were upregulated following hyperoside treatment. We note that the homologs that are highly expressed in seeds are different from those expressed in flowers, with AePAL2, AeCHS4, AeCHI3, AeFLS1, and AeUF3GaT1 being seed-specific (Figure 3B). In our previous study, we established that hyperoside promoted both flowering and seed set. Therefore, we hypothesized that hyperoside could be used as a growth regulator to stimulate pollen development in okra.

To explore how the unique flavonoid hyperoside promoting seed set in-depth mechanisms we divided reproduction into four phases from pollination to seed maturity: pollen germination (Phase I), pollen tube growth (Phase II), double fertilization (Phase III), and seed development (Phase IV; Figure 4G). Many genes participated in these processes; for example, AeGEF2 functioned as a signal molecule switch that played a similar critical role in the growth of polar cells, especially pollen germination and pollen tube growth, as reported in Arabidopsis (Chang et al., 2013). During Phase II, AeADF5 is the most prominent upregulated gene, which is consistent with our previous studies. Rapid dynamic changes of the microfilament cytoskeleton are key factors in ensuring polar growth of pollen tubes. Therefore, we examined six AeADF genes family in okra, and found that AeADF1 and AeADF5 was both significantly upregulated and AeADF5 most upregulated induced by hyperoside. According to the prediction function of AeADF5, we found that it always played important role in polymerizing microfilament. Hyperoside may play an important role in pollen tube polymerization as a signaling molecule for pollen growth and may be beneficial for vesicle transport. F-actin-related proteins also play an important role in pollen tube growth and self-incompatibility (Yang et al., 2018). During Phase III, a similar conclusion was reached that hyperoside promoted pollen growth by inducing AeFER6 expression. In Arabidopsis, the fer mutant undergoes normal female gametophyte differentiation, but about 50% of ovules are not fertilized, which is caused by a failure of pollen tube rupture (Huck et al., 2003). AeUF3GaT1 is the last and vital enzymatic step in hyperoside biosynthesis (Wang et al., 2019). That application of hyperoside leads to the maximum increase in AeUF3GaT1 transcript levels suggests a positive feedback between hyperoside treatment and AeUF3GaT1; this is consistent with the previous okra flowering study (Yang et al., 2020).

Therefore, it is important to explore whether the mechanism by which AeUF3GaT1 improving seed hyperoside content and seed set regulation is the same as the mechanism by which AeUF3GaT1 affects some aspect of okra flowering examined previously with our earlier work in okra flowers. We showed here that the key transcription factor AeMYB30 also binds to the promoter of AeUF3GaT1 in seeds, just as it did in flowers. Therefore, AeMYB30 is a universal positive regulator that responds to the application of hyperoside on reproductive tissue and organs. We also demonstrated, using AeMYB30 and AeUF3GaT1 transgenic lines that AeMYB30 enhances the transcription of AeUFG3aT1 and results in the accumulation of hyperoside in transgenic okra seeds, thereby improving reproductive development and seed set (Figures 5, C–F and 6). It broadened the mechanism of the MYB–UFGT complex in flavonol synthesis and further clarified the special role of the complex in reproductive development.

The role of plant seeds is for reproduction, but they can also provide valuable food materials full of secondary products. Flavonoids are secondary metabolites that are present at high levels in most plant seeds and grains. The flavonoids produced by seeds are diverse, and hyperoside, a main flavonol, may be specific in okra, but other flavonols and flavonoid-related compounds also play different roles in other plant seeds. Flavonoids, including flavonols, flavanols, anthocyanins, and PAs, played various roles at different stages of seed development. For example, rutin, an important class of flavonol, was maintained at high levels in buckwheat seeds, which gave them a strong bitter taste, but can effectively protect the seeds from being eaten by animals (Suzuki et al., 2015). PAs in the seed coat often possess antitoxin properties and play a defense role against fungi during seed development in Abutilon theophrasti (Paszkowski and Kremer, 1988). In the endodermis of Arabidopsis thaliana seeds, PAs accumulate in vacuoles and are oxidized in the late stage of seed development, which makes the seeds brown, accelerates the maturation and development of seeds, and causes seedlings to quickly form adult trees (Debeaujon et al., 2003).

Plant regeneration from somatic embryogenesis is a long process that depends on the genotype of the plant. The establishment of an efficient transformation system based on embryogenic callus regeneration is therefore an effective method for characterizing gene function. Compared with seedlings as starting material, embryogenic calli are more stable and have a higher regeneration potential and transformation efficiency (Ratjens et al., 2018). This embryogenic callus transformation system could be used in genome editing approaches and other nonmodel systems of high economic value in the absence of an established trans-genesis procedure. At present, many industrial plant research methods are based on mining databases for the model plants Arabidopsis, tobacco, tomato, and rice for gene prediction and then carrying out functional verification in model plants rather than the plant species in question. A stable expression system in okra is a critical step forward and was used here to confirm the roles of AeMYB30 and AeUF3GaT1 in seed development. The same procedure may be applied to any gene and will provide a foundation for research on the function of other selected genes and how they participate in the signaling pathway induced by hyperoside in okra.

Taken together, our data indicate that hyperoside, a flavonol in okra, promotes seed set by increasing the expression of AeUF3GaT1, which encodes the last enzymatic step in flavonoid biosynthesis, by the transcription factor AeMYB30. This positive effect on AeUF3GaT1 expression is caused by both endogenous hyperoside (based on our analysis of the AeUF3GaT1 overexpression lines) and exogenously supplied hyperoside (based on the hyperoside application group). In both cases, nine reproductive development-related genes affecting pollen germination, pollen tube growth, double fertilization, and seed development showed an increase in transcript levels (Figure 8).

Figure 8.

Working model of hyperoside as a unique flavonol that promotes reproductive development and increased seed set by the positive regulator AeUF3GaT1 in okra. Exogenous hyperoside application could significantly increase the expression level of AeUF3GaT1. AeUF3GaT1 efficiently improves the seed set rate by positively upregulating nine reproductive development-related genes, resulting in the promotion of pollen germination, pollen tube growth, double fertilization, and seed development. Moreover, hyperoside partially compensates for the decrease of AeUF3GaT1 from pollen germination to seed development, which indicates the relationship between hyperoside and AeUF3GaT1.

Conclusion

The exogenous application of the flavonoid hyperoside improved reproductive development and seed set in okra. AeUF3GaT1, encoding the last enzymatic step in hyperoside biosynthesis, being the most strongly induced by hyperoside application. Base on the earlier work, we uncovered a positive regulatory relationship between the transcription factor AeMYB30 and AeUF3GaT1, and it was also found AeMYB30 increases AeUF3GaT1 transcription and improves reproductive development, seed set, and hyperoside accumulation in transgenic okra seeds in this study. Taken together, our study indicates that a simple hyperoside application may enhance seed set and seed quality and thereby improve nutritional qualities in okra.

Materials and methods

Plant material and growth conditions

Seeds from the okra cultivar “Golden Glory” were sown and seedlings allowed to grow in a greenhouse for 20 d in April 2018, and then transplanted to field conditions in northern China (longitude 116.30; latitude 39.95). Fruit is generally harvested DDA16. Pollen, pistil, seeds, and other tissues were collected and stored at –20°C until used.

Hyperoside application

We added different concentrations of hyperoside (5 mg·L^–1, 10 mg·L^–1, 20 mg·L^–1, 50 mg L^–1, and 100 mg·L^–1) to buffer (inorganic salt ionic solution: 0.19 g·L^–1 KNO₃; 1.65 g·L^–1 NH₄NO₃; 0.17 g·L^–1 KH₄PO₄; 0.00178 g·L^–1 FeSO₄·7H₂O; 0.0373 g·L^–1${\text{Na}}_2^{\mathbf{\text{.}}}$EDTA; 0.004 g·L^–1 glycine; 0.0002 g·L^–1 VB ammonium sulfate hydrochloride; 0.001 g·L^–1 VB6 pyridoxine hydrochloride; 0.1 g·L^–1 inositol; 0.0062 g·L^–1 H₃BO₃; 0.00083 g·L^–1 KI), 1% (w/v) sucrose; pH 5.1) to generate stock solutions. Our previous study established that 50 mg·L^–1 (around 0.1-mM concentration) hyperoside, sprayed once every other day over 8 d during the vegetative growth stage, was most effective to induce reproductive growth. We used 1 L of solution to spray 10 plants subjected to the same growth conditions once they reached a height of ∼1 m and a crown width of 50 cm. Plants were sprayed to dripping once every other day over 8 d. Controls consisted of (1) no application and (2) spraying buffer only. Spraying started 8 d after anthesis.

Determination of total flavonoid content in seeds

We combined a mixture of 1 mL of seed extract with 0.3 mL of 5% sodium nitrite for 6 min. We then added 0.3 mL of 10% aluminum nitrate and allowed the mixture to stand for 6 min before adding 4 mL of 4% sodium hydroxide. After 15-min incubation at room temperature, we adjusted the final volume to 10 mL with 68% ethanol and measured the absorbance of the reaction mixture at 510 nm with an Agilent 1260 liquid chromatography system equipped with a Luna C18 column (250 × 4.6 mm i.d., 5 μm, Phenomenex, USA). We used 1 mL of 68% ethanol as a blank.

Extraction of total protein in seeds

We first crushed seeds just before adding liquid nitrogen and ground them under liquid nitrogen to a fine powder. We then added extraction buffer (2 mL for five seeds) and a few drops of acid to remove impurities. We then passed the seed slurry through four layers of muslin, collected the flow-through, and re-homogenized the solid residue left on muslin with 2 mL of extraction buffer. We passed the slurry through the same four layers of muslin and pooled both flow-through in the same tube. After adjusting the volume up to 5 mL with extraction buffer, we centrifuged the protein extracts at 14,000 r.p.m. at 4°C for 30 min. We discarded the pellet and determined protein concentration of the supernatant by Bradford assay.

Determination of total fat in seeds

Briefly, we dried 10 g okra seeds and crushed them before adding 200 mL N-hexane. We placed the resulting extract in a Soxhlet extractor for 6 h at 40–60°C, at a rate of 2–3 drops per second. We then removed the solvent by placing the extract in a rotating evaporator and then stored the dry powder at –20°C in the dark. We used a gas chromatograph (Agilent 7890A series) with an AT-FFA chromatographic column (30 m × 320 mm × 0.33 μm) for the identification of total seed fat. We used optimized conditions as follows: injector temperature at 260°C, split ratio of 30:1, carrier gas flow rate of 5 mL·min^–1(N₂), constant current mode; flame ionization detector temperature at 260°C, H₂O flow rate of 25 mL·min^–1, airflow rate of 300 mL·min^–1, make-up gas flow rate of 20 mL·min^–1 (N₂). We followed the following column temperature program: initial temperature of 170°C, then raised to 230°C at a rate of 20°C·min^–1 and maintained for 25 min. All reagents used in the test were analytically pure and purchased from Sigma.

Determination of total polysaccharide in seeds

We used 10 g dried okra seeds and water as solvent, heat-refluxed at 100°C for 4 h. We combined the filtrates and concentrated them by evaporation, before adding ethanol (v:v = 1:3) and centrifugation at 4,000 rpm. We discarded the supernatant and resuspended the precipitate in H₂O before dialysis for 48 h (membrane MW cut-off: 8,000–14,400). Afterward, we centrifuged the samples again at 4,000 r.p.m and then dried the supernatant to obtain a polysaccharide powder. We dissolved the crude powder in H₂O and passed it through a SephadexHW column at 40°C before freeze-drying each sample to a yellow powder. We used a spectrophotometer for detection analysis and water as a blank control.

Detection of six flavonols and three other flavonoid-related compounds: rutin, isoquercitrin, hyperoside, myricetin, quercetin-3-O-glucoside, quercetin, PAs, genistins belonging to isoflavone family, and luteolin belonging to flavone family

We started with 10 g dried okra seeds. We analyzed the nine flavonoid compounds by liquid chromatography (Agilent 1260 liquid chromatography system equipped with a Luna C18 column; 250 × 4.6 mm i.d., 5 μm, Phenomenex, USA), with the detection wavelength set to 254 nm. We used reference standards for each of the nine flavonoids (Sichuan Victor Industrial Co., Ltd., China) to quantify their seed content. The elution solvents were composed of mobile phase A (0.1% formic acid solution) and mobile phase B (acetonitrile). The gradient elution conditions were as follows: 0–45 min, 15%–25% B; 45–60 min, 25%–35%. The total run time was 60 min and the flow rate was constant at 1 mL·min^–1. The injection volume of all samples was 20 μL, and the column temperature was maintained at 35°C. The standard product information of the six flavonoids we used can be found on the website of Weikeqi Biotech Co., Ltd. (http://www.weikeqi-biotech.com/). The CAS numbers are: 1. Rutin: 153-18-4; 2. Isoquercetin: 482-35-9; 3. Hyperoside: 482-36-0; 4. Myricetin: 529-44-2; 5. Quercetin-3-O-glucoside: 21637-25-2; 6. Quercetin: 117-39-5.

SEM analysis

We used an S-3400N II scanning electron microscope (Hitachi, Japan) to analyze the samples. Dried plant material was sputtered with gold plating for 5 min to ensure an even coverage with gold. We then used double-sided conductive tape to keep samples on the sample clamp. The analysis conditions were as follows: accelerating voltage of SEM was 3.0 kV and the working temperature was 25°C.

Pollen germination and pollen tube length assay

We collected blooming flowers of okra at 10 a.m. when the flowers were in full bloom and gently removed anthers before placing them in a ventilated and cool place. The anthers were then made into powder at room temperature for 24 h to obtain dried pollen. Dried pollen was then hydrated at 25°C and 40% humidity for 10 min in pollen germination and culture medium (GM: 200 g·L^–1 sucrose, 10 mg·L^–1 CaCl₂, and 10 mg·L^–1 H₃BO₃). We then added one of six flavonoid standards (hyperoside, rutin, isoquercitrin, myricetin, quercetin-3-O-glucoside, or quercetin) at a concentration of 50 mg·L^–1 (= 0.1 mM) to GM and scored the pollen germination rate after 30 min. We measured the average pollen tube length after 1 h of cultivation, as described by Meng et al. (2018a).

RNA extraction, cDNA synthesis, and RT-qPCR

We extracted all RNA samples using the CTAB method as described by Yang et al. (2018). After incubation with RQ1 DNase (Promega) and removal of the nuclease by precipitation, we quantified total RNA using a NanoDrop spectrophotometer and confirmed RNA integrity by electrophoresis on agarose gel. One microgram of total RNA was reverse-transcribed to first-strand cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). We performed reverse transcription quantitative PCR (RT-qPCR) on the cDNAs using an Icycler iQ5 (Bio-Rad) real-time PCR system (Applied Biosystems) following the manufacturer’s instructions and using SYBR Premix ExTaq (Perfect Real Time, Takara Bio). We conducted RT-qPCR in three biological triplicates independently. Samples were normalized to actin as an internal control. Transcript levels were assessed using the 2^−ΔΔCt method. The primers used for RT-qPCR analysis are listed in Supplemental Table S1.

Vector construction and agrobacterium-mediated infiltration

To overexpress AeMYB30 and AeUF3GaT1 in okra, we ligated the coding sequence of AeMYB30 and AeUF3GaT1 and GFP into the pROKII vector to generate the overexpression vector pROKII-AeMYB30-GFP-OE pROKII-AeUF3GaT1-GFP-OE. We also generated a construct to induce RNA-interference of AeMYB30 and AeUF3GaT1 expression using the pROKII vector construct driven by the 35S promoter. We cloned a 253-bp AeMYB30 and 268-bp AeUF3GaT1 fragment into the NcoI/SwaI and Xbal/BamHI restriction site of the altered pROKII vector binary vector to generate the RNA-interference vector pROKII-AeMYB30-RNAi pROKII-AeUF3GaT1-RNAi. Recombinant plasmids were transformed into Agrobacterium (Agrobacterium tumefaciens) strain K599 in bacterial culture medium (YEP liquid medium supplemented 25-mg·L⁻¹ rifampin and 50-mg·L⁻¹ kanamycin), and the culture was shaken on a rocking platform at 180 r.p.m. for ∼12 h). Primers are listed in Supplemental Table 1.

Production and analysis of stable transformants

We used mature seeds as ex-plants for infection with Agrobacterium strains carrying the binary plasmids. We cut the seeds with a knife before setting up co-cultivation with Agrobacterium strains. Seeds and Agrobacteria were cultured together in light–dark cycles (16L: 8D photoperiod) at 25°C for 10–15 d on callus induction medium (4.43-g·L⁻¹ MS powder, 5-mg·L⁻¹ 2,4-D, 0.5-mg·L⁻¹ ZT, 500 mg/proline, 500-mg·L⁻¹ glutamine). We then transferred mature calli to bud induction medium (4.43-g·L⁻¹ MS powder, 0.5-mg·L⁻¹ NAA, 0.5-mg·L⁻¹ ZT, 500 mg/proline, 500-mg·L⁻¹ glutamine) to induce the formation of shoots. We transferred ∼3–5-cm buds to root induction medium (4.43-g·L⁻¹ MS powder, 0.1-mg·L⁻¹ IBA, 0.1-mg·L⁻¹ NAA, 500 mg/proline, 500-mg·L⁻¹ glutamine) and allowed them to grow for 20–25 d in light–dark cycles (16L: 8D photoperiod). We transferred rooted buds to the greenhouse (average temperature 25°C) and allowed them to grow for 3 months to set seeds.

Accession numbers

For UF3GaT members in Arabidopsis (https://www.arabidopsis.org/): At1G22360.1, At1G22340.1, AT1G22380.1, At1G22370.2, At2G31790.1, At2G31780.1, At1G05680.1, At5G38040.1, At1G30530.1, At1G22400.1, At5G17050.1, At5G17030.1, At5G17040.1, and At1G30540.2. For okra genes (https://www.ncbi.nlm.nih.gov/genome/14290?genome_assembly_id = 323476): AePAL1(LOCKP006666), AePAL2 (LOCKY635876), AePAL3 (LOCKJ606999), AeC4H (LOCKC404826), Ae4CL1 (LOCKC252632), Ae4CL2 (LOCKJ607007), Ae4CL3 (LOCKJ607008), Ae4CL4 (LOCKJ607009), AeCHS1 (LOCKF246682), AeCHS2 (LOCJN114733), AeCHS3 (LOCKJ606990), AeCHS4 (LOCMF668672), AeCHI1 (LOCMF348726), AeCHI2 (LOCKJ607006), AeCHI3 (LOCKC488172), AeF3H1 (LOCMG372370), AeF3H2 (LOCMG372371), AeF3H3 (LOCMG792349), AeF3H4 (LOCMG372369), AeF3H5 (LOCKP222315), AeFLS1 (LOCKJ606996), AeFLS2 (LOCKX257998), AeFLS3 (LOCMF668660), AeFLS4 (LOCKP222367), AeFLS5 (LOCKF514665), AeF3’H1 (LOCMF668666), AeF3’H2 (LOCMF668637), AeF3’5’H1 (LOCKJ607000), AeF3’5’H2 (LOCJN114933), AeF3’5’H3 (LOCJN114850), AeUF3GaT1 (LOCMF668661), AeUF3GaT2 (LOCMF668662), AeUF3GaT3 (LOCMF668666), AeUF3GaT4 (LOCMF668668), AeUF3GaT5 (LOCKX257998), AeUF3GaT6 (LOCMF770708), AeUMYB30 (LOCMF668663), AeROP1 (LOCKY635876), AeROP2 (LOCKJ607004), AeGEF1 (LOCMT316193), AeGEF2 (LOCMT316192), AeRLK6 (LOCMG372369), AeLRX1 (LOCKX257999), AeLRX11 (LOCMF668649), AeADF5 (LOCMG372369), AeSCAB1 (LOCMF668675), AePEMI1.2(LOCKC252632).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. The effect of rutin on okra fruit size, seed set rate, and other physiological indexes.

Supplemental Figure S2. The effect of isoquercetin on okra fruit size, seed set rate, and other physiological indexes.

Supplemental Figure S3. The effect of myricetin on okra fruit size, seed set rate, and other physiological indexes.

Supplemental Figure S4. The effect of quercetin-3-O-glucoside on okra fruit size, seed set rate, and other physiological indexes.

Supplemental Figure S5. The effect of quercetin application has no obvious effect on okra fruit size, seed set rate, and other physiological indexes.

Supplemental Figure S6. The effect of hyperoside on fruit morphology.

Supplemental Figure S7. The effect of hyperoside on seeds 16 d after anthesis.

Supplemental Figure S8. The expression of genes for key enzymes in the phenylpropanoid pathway, including flavones, isoflavonoid, flavonol, and proanthocyanidins biosynthetic pathway induced by hyperoside.

Supplemental Figure S9. Phylogenetic tree analysis of AeUF3GaT1 and AtUF3GaTs.

Supplemental Figure S10. Amino acid sequence alignment of AeUF3GaT1 and AtUF3GaT1.

Supplemental Figure S11. The relative expression of five representative genes of phase IV 0, 2, 4, 8, 1, 16, and 20 d after anthesis.

Supplemental Figure S12. Callus to whole-plant regeneration.

Supplemental Figure S13. The relative expression of six representative genes of phases I, II, III, and IV in WT, empty vector, AeMYB30-RNAi-line1, AeMYB30-RNAi-line2, AeMYB30-OE-line1, AeMYB30-OE-line1, AeUF3GaT1-RNAi-line1, AeUF3GaT1-RNAi-line2, AeUF3GaT1-OE-line1, and AeUF3GaT1-OE-line2 in control, buffer, and hyperoside application three groups.

Supplemental Table 1. Primers used in this study.

 

Y.F., Q.Y., and D.M. designed this project. Q.Y. and D.M. performed most of the experiments, and Q.Y. wrote the manuscript. Z.S. performed the qRT-PCR analysis. B.D. and Z.S. provided the plant materials. L.N. and H.L. measured the hyperoside content. T.D., T.L., H.C., and W.Y. analyzed the data and we all discussed the article with Y.F.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys) is: Dong Meng (mengdongjlf@163.com), Yujie Fu (yujie_fu@163.com).